The separation process effected through high performance liquid chromatography relies on the fact that a number of component solute molecules in a sample stream of a fluid (known as the mobile phase) flowing through a packed bed of particles (known as the stationary phase) usually in a column, can be efficiently separated from one another. Basically, because each individual sample component has a different affinity for the stationary phase, each such component has a different rate of migration through and a different exit time from the column, effecting separation of the components. The separation efficiency is determined by the amount of spreading of the solute band as it traverses the bed or column.
Such separations, particularly in preparative processes, have significant limitations typically occasioned by the batch nature of the processes. Typically, a chromatographic column is first equilibrated by flowing an equilibrating fluid through the column, the latter is then charged or loaded with a fluid mixture containing the solute or solutes sought to be separated, and one or more eluant fluids are flowed sequentially through the column to release bound solute selectively. The eluted solutes are thus temporally separated in the flowstream emerging at the output of the column and the process may be repeated cyclically. At typical flow rates and where the concentration of the desired solutes in the initial fluid mixture are low, the production of significant quantities of the desired solutes can be an unhappily slow and very expensive process with current apparatus and methods that require comparatively long cycle times.
A chromatography system of the prior art, as shown schematically in FIG. 1, typically includes chromatographic column 20 the input of which is usually fed from a plurality of reservoirs provided for storing at least a corresponding plurality of different fluids. Thus, reservoir 22 serves to store a supply of equilibrating fluid 24, while reservoirs 28 and 26 respectively store corresponding supplies of eluant fluid 32 and fluid mixture 30 containing the sample to be separated. Pump 34 is usually coupled between the proximal or input end of column 20 and a plurality of valves means 36, 37 and 38 respectively connected to the outputs of reservoirs 22, 26 and 28, pump 34 serving to force fluid from the various reservoirs into column 20. Disposed adjacent the distal or output end of column 20 is the usual detector 39 of any type well known in the prior art.
In operation, column 20 is usually first equilibrated by opening valve means 36 and running pump 34 so as to permit fluid from reservoir 22 to be pumped at a predetermined flow rate through column 20 to equilibrate the latter. Then, typically valve means 36 is closed and valve means 37 is opened to permit a quantity of fluid mixture 30 to be pumped into column 20, loading the latter with solute molecules that bind to chromatographically reactive surfaces located within column 20. Lastly, valve means 36 and 37 are closed and valve means 38 is opened to permit eluant fluid 32 from reservoir 28 to be pumped through the loaded column. The solute molecules elited from the column by the eluant fluid are detected, typically optically by detector 39, and may be separated in a known type of fraction collector 35.
The use of several reservoirs for the equilibrating fluids and eluants, all being fed alternatively into the column by a pump through extensive conduit and valve systems, leads to mixing of the various fluids, contributes to band spreading, is wasteful of often expensive eluant fluid, and introduces undesirable delays in operation occasioned by valve switching and the necessity of transferring the volume of unexpended fluids temporarily stored within the conduits and valves. Such delays until very recently have been considered negligible compared to the relatively long cycle time in the column. There is also the inevitable mixing that occurs when the various fluids are introduced into and expelled from the same pump, both the delays and the mixing tending to promote band-broadening and reduce efficiency. The time required ordinarily between the introduction of sample into the column and the ultimate separation of the sample components at the column output can readily exceed many hours and often days.
A major problem impeding speed and throughput of separations in prior art HPLC systems arises out of the use of chromatography columns operating under the constraints imposed by the well-known Van Deemter equations and the consequent arrangement of the physical components of the system.
Because chromatographic system obeying the Van Deemter equations are believed to operate with substantially laminar fluid flow, the fluid wave fronts of the different fluid flows into the column tend to assume paraboloidal configurations, thereby precluding sharp separations between volumes of different fluids traversing the column, contributing to greater mixing. It has recently been discovered that the limitations imposed on such HPLC separations by operation at a mobile phase flow rate dictated as optimal by the Van Deemter curves can be overcome by novel methods of performing liquid chromatography employing an eluant flow rate through the chromatography column at a speed corresponding to an average reduced velocity (as hereinafter defined) greater than about 5000. It is believed that under such conditions, turbulent flow of the eluant is induced within the column and it is postulated that such turbulent flow enhances the rate of mass transfer, thus increasing the throughput/productivity of the column by reducing dramatically the time required to effect separations. These novel methods of and apparatus for performing liquid chromatography are described more fully in U.S. patent application Ser. No. 08/552193 filed Nov. 2, 1995, the same being incorporated in its entirety herein by reference.
It has been customary to describe the function of an HPLC column in a plot in which column plate height H is plotted against linear velocity u of the mobile phase. Since an HPLC process is a diffusion-driven process and since different solute molecules have different diffusion coefficients, one can consider this latter variable in applying the process to a wide range of solutes of different molecular weights. Additionally, the size of the particles in the column may differ from column to column, and may also be considered as another variable. Similarly, the viscosity of the solvent for the solute might be considered. In order to normalize the plots to take these variables into account, one advantageously may employ reduced coordinates, specifically, h in place of H, and v in place of u, as taught by Giddings and described in Snyder & Kirkland Introduction to Modern Liquid Chromatography, 2nd Ed., John Wiley & Sons, Inc., (1979) at pp. 234-235, to yield a reduced form of the Van Deemter equation as follows: EQU h=a+b/v+cv, or (Equ. 1) EQU H=ad.sub.p +bD/u+cud.sub.p /D (Equ. 2)
wherein a, b and c are coefficients, and the coordinate h is defined as H/d.sub.p, d.sub.p being the particle diameter; accordingly h is a dimensionless coordinate. Similarly, the dimensionless coordinate, the reduced velocity v, is defined as ud.sub.p /D where D is the diffusion coefficient of the solute in the mobile phase.
It will be recognized that v is also known as the Peclet number. It should be stressed, however, that the reduced coordinate or Peclet number, v, as used in the instant exposition of the present invention, is descriptive of fluid flow through the entire column, and should not be considered as descriptive of fluid flow within the pores of porous particles that may constitute a packed bed in the column.